The phosphoryl group on the active-site His of enzyme I is transferred to the active-site His residue of Hpr, and it has been proposed that the major interaction between enzyme I and HPr occurs via the alpha- helical subdomain in the amino-terminal domain of enzyme I. The isolated recombinant alpha-helical domain (residues 25-145) with an estimated 80 percent alpha-helices by far-UV circular dichroism (CD) spectra as well as enzyme I deficient in that domain [EI(minus HD)] from M. capricolum with an estimated 50 per cent alpha-helical content by CD were used in isothermal titration calorimetry (ITC) experiments with HPr. We found that HPr binds to the alpha-helical domain and intact enzyme I with apparent association constants of 50,000 and 140,000/M at pH 7.5 and 25 C, respectively, but HPr did not bind to the deletion mutant EI(minus HD). This is consistent with the alpha-helical domain of enzyme I being necessary for its interaction with HPr. However, intact enzyme I and EI(mnus HD) both dimerized in sedimentation equilibrium experiments; data analysis gave log K values for dimerization of 5.0 and 6.7, respectively, at pH 7.5 and either 4 or 20 C. Moreover, the C-terminal domain of enzyme I (38,027 Da with a His tag composed of Met plus 6-His) was found to strongly self- associate with a log K value of about 10. Thus, the N-terminal domain of enzyme I exerts a negative effect on enzyme I dimerization and thereby has a potential regulatory role in the autophosphorylation reaction of enzyme I with PEP, which has been thought to require the dimer structure. It remains to be proven whether phosphorylation of the active-site His in the N-terminal domain also affects the degree of dimerization of enzyme I under physiological conditions. - PEP:sugar phosphotransferase system, phosphoryl transfer, enzyme I N- and C- terminal domains, alpha helical deletion mutant of enzyme I